生长抑制因子4基因表达对K562细胞生长的影响

目的 研究生长抑制因子4（ING4）基因表达对K562细胞增殖的影响。方法 将经过定点突变技术人源化改造的ING4（鼠→人）生长抑制因子4基因酶切并连接到pAdTrack-CMV转移质粒上，然后与腺病毒质粒pAdEasy-1共转化BJ5183细菌，获得重组腺病毒质粒pAdEasy-1-pAdTrack—CMV—hING4，将它转染QBI-293A细胞后收获重组腺病毒Ad—hING4（以下简称为Ad-ING4）。将Ad-ING4感染K562细胞，光学显微镜下观察病毒感染前后细胞形态的变化，免疫细胞化学分析凋亡因子的改变，激光扫描共聚焦显微镜观察K562细胞的凋亡，流式细胞术（FCM）检测细胞凋亡率。结果 成功构建了人ING4腺病毒质粒，获得高滴度的重组腺病毒Ad-ING4，用它感染K562细胞72h后，FCM法测定细胞凋亡率为19．7％，与空病毒对照组相比差异有统计学意义（P〈0．01），ING4基因能下调K562细胞bcl-2的表达并上调bax的表达，多种方法检测结果表明ING4基因能抑制K562细胞生长，诱导凋亡。结论 重组腺病毒Ad—ING4可以显著抑制K562细胞的生长。

Ad-ING4 inhibits K562 cell growth

OBJECTIVE: To observe the effect of recombinant adenovirus Ad-ING4 on K562 cells. METHODS: Human ING4 recombinant transfer vector pAdTrack-CMV-ING4 was constructed by enzyme digest and ligation of human ING4 gene which was obtained through site specific point mutation of mouse ING4. The vector was co-transduced into BJ5183 E. coli with pAdEasy-1. The new recombinant adenovirus vector pAdEasy-1-pAdTrack-CMV-hING4 was transfected into QBI-293A cells. To obtain the ING4 recombined adenovirus (Ad-ING4). Ad-ING4 was used to infect K562 cells. The effect on K562 cells of ING4 was tested by LSCM FCM and immunohistochemistry. RESULTS: Human ING4 recombinant adenovirus vector was constructed successfully, and high titre ING4 recombinant adenovirus (Ad-ING4) was obtained. ING4 can down-regulate the expression of bcl-2 and up-regulate expression of bax. The apoptosis of K562 cells induced by ING4 was proved by LSCM FCM and immunohistochemistry. The apoptosis rate was 19.7% (after 72h), which displayed significant difference compared with that of control groups (P < 0.01). CONCLUSION: Ad-ING4 can inhibit the growth of K562 cells and induce the cells apoptosis. The human ING4 recombinant adenoviral vector constructed might provide an approach to the target therapy of tumors.